Combined Effects of Confinement and Macromolecular Crowding on Protein Stability.

Murial L. Ross,Steven Long,Prashanth Asuri,Jeffrey Kunkel

doi:10.3390/ijms21228516

Murial L. Ross, Steven Long + Show 2 more

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https://doi.org/10.3390/ijms21228516

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Abstract

Confinement and crowding have been shown to affect protein fates, including folding, functional stability, and their interactions with self and other proteins. Using both theoretical and experimental studies, researchers have established the independent effects of confinement or crowding, but only a few studies have explored their effects in combination; therefore, their combined impact on protein fates is still relatively unknown. Here, we investigated the combined effects of confinement and crowding on protein stability using the pores of agarose hydrogels as a confining agent and the biopolymer, dextran, as a crowding agent. The addition of dextran further stabilized the enzymes encapsulated in agarose; moreover, the observed increases in enhancements (due to the addition of dextran) exceeded the sum of the individual enhancements due to confinement and crowding. These results suggest that even though confinement and crowding may behave differently in how they influence protein fates, these conditions may be combined to provide synergistic benefits for protein stabilization. In summary, our study demonstrated the successful use of polymer-based platforms to advance our understanding of how in vivo like environments impact protein function and structure.

Highlights

Until recently, most in vitro investigations of protein function and stability have been based on simple buffer systems that do not closely mimic the complex in vivo cellular environments [1,2,3,4]
The latter outcome is significant because confinement and crowding are often used synonymously, even though these environments are distinct in how they exert their influence on protein fates
We describe our attempts to build on previous research and develop conditions to study the combined effects of confinement and crowding on protein stability [17,24,25,26]

Summary

Introduction

Most in vitro investigations of protein function and stability have been based on simple buffer systems that do not closely mimic the complex in vivo cellular environments [1,2,3,4]. Several studies have proposed the use of high concentrations of natural and synthetic macromolecules to study crowding [7,8,9,10], and encapsulating proteins within the pores of silica, polyacrylamide, or other hydrogels to study confinement [11,12,13,14,15,16,17] These studies have contributed to the mechanistic understanding of how proteins behave in vivo, as well as to unravel the differences between how confinement and crowding influence protein function [14,16,17]. Both theoretical and experimental, have demonstrated that this distinction is important in how the two environments may affect protein structure and function [14,18,19]

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